While the inhibitory neurotransmitters glycine and GABA are co-released at many auditory brainstem synapses, exactly how these transmitters are co-released, and the physiological function of this co-transmission remain ambiguous. In the dorsal cochlear nucleus (DCN), inhibition from glycine/GABA co-releasing interneurons termed cartwheel cells tightly controls whether target neurons fire in response to multi-sensory inputs. Thus, a lack of knowledge as to the cellular mechanisms governing the amount of inhibitory transmitters in synaptic vesicles severely limits our understanding of DCN circuit function. The objective of this proposal is to determine the mechanisms that regulate the differential packaging of glycine and GABA in pre-synaptic vesicles, and establish how co-release dictates the temporal kinetics of post-synaptic inhibition. Aim 1 will employ dual whole-cell recordings in connected pairs of cartwheel cells to determine the extent to which the membrane glycine transporter GlyT2 regulates the ratio of glycine/GABA in synaptic vesicles. Aim 2 will experimentally manipulate pre-synaptic glycine/GABA ratios in cartwheel cell pairs to test whether co-release functions to sharpen the deactivation kinetics of post-synaptic receptors. Aim 3 will use 2-photon Na+ imaging to determine whether GlyT2 expression is functionally homogenous across multiple boutons from the same cell, or if there exists significant variability in the sub-cellular distribution of glycine transporters. These experiments will shed light on the fundamental mechanisms that regulate inhibitory co-transmission, and establish how this shapes the temporal properties of local inhibition in the DCN.